Process of extracting antidiabetic substance from pancreas



Patented Sept. 14, 1948 PROCESS OF EXTRACTING ANTIDIABETIC SUBSTANCEFROM PAN CREAS Carl Ludwig Lautenschlager and Fritz Lindner,Frankfort-on-the-Main, Germany; vested in the Attorney General of theUnited States No Drawing. Application March 25, 1943, Serial No.480,595. In Germany January 29, 1938 '1 Claim. 1

This invention is a continuation-in-part of our U. S. application SerialNo. 253,340, filed January 28, 1939, now abandoned for: Blood sugarlowering substance obtained from the pancreas and a process of preparingit in the name of Carl Ludwig Lautenschlager and Fritz Lindner.

The present invention relates to an anti-diabetic product which can beisolated from the pancreas of animals and is useful as an antidiabeticagent, particularly in that it possesses 1a prlotracted action inlowering the blood sugar eve.

The isolated anti-diabetic component of the pancreas, known as insulin,is a well defined albuminous substance crystallizable in the form of itszinc salt, and by virtue of its molecular weight it has been classifiedas an albuminose. The properties of this product have been exactlydetermined. For instance, it has quite a sharp iso-electrical point at apH-value of between about 5.3 to about 5.5, which, however, to a certainextent is determined by the nature of accompanying substances. Below apH-value of l and above a pI-I-value of 6, this form of the isolatedhormone is readily soluble in water.

A large number of processes are known for the isolation of theanti-diabetic substance from the pancreas. The operating conditionsemployed in these processes are based on the discovery of Best andBanting that the hormone is, rather, sensitive to chemical and physicalaction, particularly to the influence of albumen-splitting fermentswhich are present in the pancreas besides the hormone. It was more orless clearly intended that the hormone should be obtained by theseprocesses in a form in which it is as far as possible freed fromaccompanying substances, since it was generally assumed that ananti-diabetic agent of particularly high eflicacy could be obtained onlyin this manner.

In distinction from this, it is an object of the present invention toisolate the hormone or antidiabetic substance more nearly in the form inwhich it is present in the pancreas. We have found that the producthitherto known as insulin does not constitute the native form of thehormone, but is a cleavage product of a true complex compound present inthe pancreas. We were, therefore, confronted with the problem ofisolating a product which would more closely resemble the natural formof the blood sugar lowering agent in the pancreas and to determine whatits physical eifect might be.

We have discovered that this object can be accomplished by applyingoperating conditions of the isolating methods which take regard to thesensitive nature of the active substance against chemical and physicalactions, even more than do the known methods. As a result, we haveobtained an active substance which possesses better solubilityproperties in an aqueous medium than the hitherto known active agent.The'new product is insoluble in water in a pH-range of between about 6to about 8, whereas, as has been pointed out, the known hormone isreadily soluble in water above a pH-value of 6. The new antidiabeticproduct is, therefore, insoluble at'the pI-I-value predominating in thehuman blood, and this property entails a retardation of the rate ofresorption of the substance upon injection in the form of an aqueoussuspension, or in other words, it causes a prolonged action of the newsusbtance. This result is obtained without the addition of anyphysiologically incompatible substance, thus establishing a particularlyuseful improvement, since the effect of each single injection per unitof time is prolonged thereby and the number of injections required mightbe reduced. Simultaneously, the action of each injection is uniformlyextended for a prolonged period, and the occasional hypoglycaemia whichusually follows theinjection of hitherto known hormone preparations doesnot occur.

The known isolation methods comprise aseries of steps beginning with thetreatment of the fresh glands for the purpose of preserving and cleaningthem, followed by extraction of the active principle from the gland andits precipitation from the extract accompanied by variousexpedientsaiming at the purification and con centration of the precipitate. Wehave found that in order to isolate the new product, it is advantageousthat these various steps be performed at low temperatures, that anyintermediate products, prior to the isolation of the final substance, beworked up rapidly, and that phase of the process which is carried out inthe alkaline nately, it may be suitable to acidify thesolution beforeremoving the precipitate formed by the addition of alkali, and then tocentrifuge the solution until it has been clarified. j

According to the present invention, the pan- 55' creas gland may beextracted in fresh condition.

However, fresh glands which have been preserved by an acid treatmentcombined with a low cooling treatment in the usual manner, may likewisebe used with the same result. This is essential for the working up ofthe glands procured from slaughter-houses which are not situated nearthe extraction plant.

Details of the new isolation methods may be gathered from the examplesgiven herein. It should be observed that it is characteristic for allvariations of our invention that the active substance must beprecipitated from .the final solution by adjusting this solution to apH-value between about 6 and about 8.

A substance precipitating at the high pllevalue just mentioned hasalready been observed in the performance of the known process for thepreparation of the hormone. This substance, however, has been found notto be an antidiabetic product, and it has a character quite differentfrom that of the active component obtainable by the present invention.It has been recognized as a ballast substance in the manufacture of thehormone of the pancreas and has been discarded. In view of the knownsensitivity of the hormone to chemical and physical actions, it has alsobeen proposed to perform the extraction and precipitation steps asmildly as possible. But an active substance having the properties hereindisclosed has never been obtained. The operating conditions employed arenew. In this connection, it should be emphasized that the invention isnot limited to the steps indicated in the present examples, but includesall variations of these steps Which may yield the same complex compound.For instance, an exact limitation as to temperatures and time periods oftreatment is impossible, since deviations from optimum conditions do notnecessarily make the new methods inoperative, but might merely result insmaller yields or in products of lesser degrees of purity. Allmodifications are within the purview of our invention, and the claimswhich read to a reactive substance which has been precipitated from thefinal solution by adjusting it to a pI-L-Valu'e bet-ween about 6 andabout 8.

In the raw state, the new product contains from 8 to 16 internationalunits per milligram, the degree of purity widely depending on the natureof the base material. After purification by precipitation the product isobtained in a condition of remarkably high uniformity and containsbetween 12 and 16 international units per milligram.

The analytic figures found for the most important constituents of thenew product in comparison with crystallized insulin are shown in thefollowing table:

It results therefrom that the new product as compared with thecrystallized insulin shows a higher content of nitrogen which isobviously due to the higher content of basic constituents, especiallarginine and lysine. In contradistinction thereto, the content ofcrystine and tyrosine 4 is considerably lower than in the case of thecrystallized insulin.

Since it was furthermore possible to obtain crystallized insulin bycareful fermentative cleavage of the new product, it derives from theseresults that the new product is a combination of the usual insulin withfurther albuminous substances of basic nature.

For the purpose of further clarification, the behaviour of the newproduct during electrophoresis was examined and the molecular weightswere determined.

By electrophoresis it was clearly found that in solutions of pH= 3.5 and4.2 the new product represents a uniform substance and that nosubstances of other molecular weight and charge are present.

This surprising fact was confirmed by the molecular weightdeterminations carried through according to the ultra-centrifuge methodas well as with the aid of osmotic pressure.

The followin examples serve to illustrate the invention, but they arenot intended to limit it thereto:

1. 20 kilos of pancreas of cattle are removed immediately afterslaughter, freed from fat and connective tissue, cut into small piecesand introduced into a mixture of 6 kilos of finely ground ice, 4 litresof water and 540 cc. of concentrated hydrochloric acid. The whole isvigourous'ly stirred until the main quantity of the ice has melted andis then frozen in a freezing mixture. The deposit is brought in a hardfrozen state to the place where it is to be further treated and is thereground in a mixture cooled to 0 C. of 54 litres of alcohol of 99.8 percent strength and 6.5 litres of water. The whole is stirred for 12 hoursat 0 C. and filtered. The residue is further stirred for 2 hours atabout 10 C. with 40 litres of alcohol, 28 litres of water and cc. ofconcentrated hydrochloric acid and the mixture is again filtered. Theextracts are cooled to 0 C. and divided into portions of 15 litres whichare standardized by means of concentrated. ammonia to a pH-value of 7.4to 7.6 and rapidl centrifuged until clear. The clear solutions are thenimmediately acidified by means of dilute sulfuric acid to a pH-value of3. The united acid solutions are concentrated under reduced pressure to10 litres. The solution is standardised to a pH-value of 2.8 and the fatis removed by extracting with ether. The clear solution is precipitatedby means of 25 per cent of sodium chloride, the precipitate is allowedto stand over night and filtered; then it is introduced into 1200 cc. ofwater and precipitated by means of 15 per cent of sodium chloride. Thesecond sodium chloride precipitate is dissolved in 2 litres of water andthe pH-value of the solution is standardized to 7.2 to 7.4. Theflocculent precipitate obtained is centrifuged immediately, washed withwater and dried with ace.- tone and ether. 4 grams of a preparation arethus obtained which contains about 15 units per milligram and has amarked prolonged action when injected in the form of an aqueoussuspension.

2. .20 kilos of glands of cattle are removed about a quarter of an hourafter slaughter, freed from fat and connective tissue and immediatelyfrozen by means of a low cooling process with a freezing liquor or bymeans of solid carbon dioxide. The further treatment is carried out asdescribed in Example 1.

20 kilos of mutton glands are removed as soon as possible afterslaughter; they are prepared and immediately ground in a mixture cooledto C. of 54 litres of alcohol, 16.5 litres of water and 540 cc. ofconcentrated hydrochloric acid. The whole is further treated asdescribed in Example 1.

4. 100 kilos of glands of cattle which have been pretreated as describedin Example 1 are ground in a mixture of 270 litres of alcohol of 99.8per cent strength and 32.5 litres of water. The extraction is performedas described in Example 1, only during the first extraction thetemperature is gradually raised to 10 C. to 15 C. The extracts obtainedare then adjusted at 10 C. to 15 C. with concentrated ammonia to a pH-value of 7.4 to 7.6 and then brought at once to a pH-value of 2.5 to 3by the addition of sulfuric acid of 50 per cent strength. The whole isthen centrifuged until a clear solution is obtained which is furthertreated as described in Example 1 until the second precipitation withsodium chloride is complete. This second sodium chloride precipitate isdissolved in 2.5 litres of water, the solution is mixed with 20 grams ofsodium chloride and then adjusted by means of N-caustic soda solution toa pH-value of 6.8 to 7. The precipitate is centrifuged and dried and thefiltrate is further treated as normal hormone. Per 1 kilo of pancreasthere are obtained about 20-25 g. of the new preparation and con taining5-10 units per milligram about 500 to 1000 units of normal hormone.

5. 100 kilos of glands of calf which were removed immediately afterslaughter and preserved at a low temperature are ground in the frozencondition in a mixture of 270 litres of alcohol of 99.8 per centstrength, 82.5 litres of water and 5.4 litres of hydrochloric acid(1:1). The further treatment until the second sodium chlorideprecipitate is obtained is the same as described in Example 4. Thissecond sodium chloride precipitate is dissolved in 6 litres of water andadjusted by means of N-ammonia to a pH-value of 7. The precipitate is atonce centrifuged and dried and the mother liquor is worked up as normalhormone. Per 1 kilo of glands there are obtained about 8000 units of thenew preparation and 2000 to 4000 units of normal hormone.

6. 100 kilos of glands of cattle which have been pretreated as describedin Example 1 are ground in a mixture of 270 litres of alcohol of 99.8per cent strength and 32.5 litres of water. The extraction is performedas described in Example 1, only during the first extraction thetemperature is gradually raised from 10 C. to C. The extracts obtainedare then adjusted at 10 C. to 15 C. with concentrated ammonia to apH-value of 7.4 to 7.6 and then brought at once to a pH-value of 2.5 to3 by the addition of sulfuric acid of 50 per cent strength. The whole isthen centrifuged until a clear solution is obtained which is furthertreated as described in Example 1 until the second precipitation withsodium chloride is complete. This second sodium chloride precipitate isdissolved in 2.5 litres of water, the solution is mixed with grams ofsodium chloride and then adjusted by means of N-caustic soda solution toa pH-value of 6.5. The

6 precipitate is centrifuged and dried and the filtrate is furthertreated as normal hormone. Per 1 kilo of pancreas there are obtainedabout 2000 to 2500 units of the new preparation and about 500 to 1000units of normal hormone.

7. 100 kilos of glands of calf which were removed immediately afterslaughter and preserved at a low temperature are ground in the frozencondition in a mixture of 270 litres of alcohol of 99.8 per centstrength, 82.5 litres of water and 5.4 litres of hydrochloric acid(1:1). The further treatment until the second sodium chlorideprecipitate is obtained is the same as described in Example 1. Thissecond sodium chloride precipitate is dissolved in 6 litres of water andthen adjusted by means of N-ammonia to a pH-value of 6.5. Theprecipitate is at once centrifuged and dried and the mother liquor isworked up as normal hormone. Per kilo of glands there are obtained about8000 units of the new preparation and 2000 to 4000 units of normalhormone.

We claim:

The process which comprises extracting the pancreas with a mixture ofalcohol and hydrochloric acid at a temperature ranging from freezing to15 C., separating the liquid extract from the remaining solid, addingammonia to the liquid extract until the pH of the liquid extract isabout 7.5, whereby a precipitate forms in the mother liquid, immediatelyseparating such precipitate from the mother liquid by rapidcentrifugating, immediately adjusting the pH of the liquid to about 3,concentrating said liquid by distillation under reduced pressure, addingto the concentrated liquid a solution of sodium chloride to cause theformation of a precipitate, separating the precipitate, dissolving thesame in water, treating the resulting solution with a weaker solution ofsodium chloride than that previously employed to produce a precipitate,dissolving said precipitate in water adjusting the solution to a pHvalue between about 6 and 8 and isolating the precipitate thus formed.

CARL LUDWIG LAU'I'ENSCHLKGER. FRITZ LINDNER.

REFERENCES CITED The following references are of record in the file ofthis patent:

UNITED STATES PATENTS Number Name Date 1,469,994 Banting et a1. Oct. 9,1923 1,520,673 Walden Dec. 23, 1924 1,547,515 Murlin July 28, 19251,626,044 Macy Apr. 26, 1927 OTHER REFERENCES Carbohydrate Metabolismand Insulin by Macleod, 1926, pages to 77.

Chemical and Physiological Properties of the Internal Secretions byDodds and Dickens, 1925, pages 7 to 26.

Chemical Abstracts, vol. 21, 1927, article by Sordelli in pp. 3252 and3253.

New 8: Nonoflicial Remedies, Jan. 1, 1938, pages 339 to 344.

